ii. chemistry 223 



2. Methods of Isolation 



a. General 



'I'he methods of isolation reported l)y the various investigators-"® de- 

 {)(Mul(>(l ui)()n the use of some of the followino; propeities of pyridoxine: (I) 

 adsorption on fuller's earth, or charcoal; (2) solul)ility in neutral ethanol 

 or acetone; (3) stability to acid and alkaline hydrolysis; (4) failure to pi'e- 

 cipitate with heavy metal salts; (o) formation of an acetyl derivative; and 

 (()) precipitation with phosphotungstic acid. The sources used were rice 

 polishings or l)ran and yeast. 



h. Kuhn and Wcndi 



The method used by Kuhn and Wendt^ involved partial purification of a 

 heat- and alkali-sensitive protein complex in freshly prepared Lebedew 

 juice from that unique source, Miincher I^owenbrau yeast. Low molecular 

 weight impurities could be dialyzed away at temperatures below 3°, leaving 

 behind the vitamin-protein complex. After further purification of the com- 

 plex as a protein, the prosthetic group of the protein was split off by heat- 

 ing. Treatment with acetic anhydride yielded a chloroform-soluble acyl 

 derivative which then was crystallized. After hydrolysis the hydrochloride 

 was obtained in crystalline form. 



c. Lepkovski/ 



Using a barium hydroxide extract of a fuller's earth adsorbate prepared 

 from rice bran extract, Lepkovsky" effected further purification of his fac- 

 tor I concentrate by removal first of alcohol-insoluble mercury salts, and 

 then of the water-insoluble lead salts followed by precipitation of the xita- 

 min with phosphotungstic acid. The phosphotungstate was then crystal- 

 lized. The vitamin was recovered by decomposing this product with barium 

 hydroxide and final crystallization of pyridoxine (or factor I, as then named 

 by this investigator) as presumably the sulfate. 



d. Gijorgy 



Peter's eluate,^** a yeast concentrate of thiamine widely used before com- 

 mercial thiamine chloride was available, served as the starting material for 

 ^'Vorgy* in his isolation of pyridoxine. From this he prepared a barium hy- 

 droxide eluate of fuller's earth adsorbate. Then this was freed of neutral 

 alcohol-ethyl acetate-insoluble impurities. Further purification was effected 



« S. Lepkovsky, J. Biol. Chem. 124, 125 (1938). 



1" H.W. Kinnersley, J. R. O'Brien, R. A. Peters, and V. Reader, Biuchcm. J. 27. 

 225 (1933). 



